General rights Copyright and moral rights for the publications
made accessible in the public portal are retained by the authors
and/or other copyright owners and it is a condition of accessing
publications that users recognise and abide by the legal
requirements associated with these rights.
Users may download and print one copy of any publication from
the public portal for the purpose of private study or research.
You may not further distribute the material or use it for any
profit-making activity or commercial gain
You may freely distribute the URL identifying the publication in
the public portal If you believe that this document breaches
copyright please contact us providing details, and we will remove
access to the work immediately and investigate your claim.
Downloaded from orbit.dtu.dk on: May 20, 2019
Dangerous relations in the Arctic marine food web: Interactions
between toxinproducing Pseudo-nitzschia diatoms and Calanus
Hardardottir, Sara; Pancic, Marina; Tammilehto, Anna; Krock,
Bernd; Mller, Eva Friis; Nielsen, TorkelGissel; Lundholm,
NinaPublished in:Marine Drugs
Link to article, DOI:10.3390/md13063809
Document VersionPublisher's PDF, also known as Version of
Link back to DTU Orbit
Citation (APA):Hardardottir, S., Pancic, M., Tammilehto, A.,
Krock, B., Mller, E. F., Nielsen, T. G., & Lundholm, N.
(2015).Dangerous relations in the Arctic marine food web:
Interactions between toxin producing Pseudo-nitzschiadiatoms and
Calanus copepodites. Marine Drugs, 13(6), 3809-3835.
Mar. Drugs 2015, 13, 3809-3835; doi:10.3390/md13063809
marine drugs ISSN 1660-3397
Dangerous Relations in the Arctic Marine Food Web: Interactions
between Toxin Producing Pseudo-nitzschia Diatoms and Calanus
Sara Harardttir 1,2,*, Marina Pani 1,2, Anna Tammilehto 1, Bernd
Krock 3, Eva Friis Mller 4, Torkel Gissel Nielsen 2 and Nina
1 Natural History Museum of Denmark, University of Copenhagen,
Slvgade 83S, 1307 Copenhagen, Denmark; E-Mails:
[email protected] (M.P.); [email protected] (A.T.);
[email protected] (N.L.)
2 National Institute of Aquatic Resources, Technical University
of Denmark, Charlottenlund Slot, Jgersborg All 1, 2920
Charlottenlund, Denmark; E-Mail: [email protected]
3 Alfred-Wegener-Institut fr Polar- und Meeresforschung,
kologische Chemie, Am Handelshafen 12, 27570 Bremerhaven, Germany;
E-Mail: [email protected]
4 Arctic Research Center, Department of Bioscience, Roskilde,
Aarhus University, Frederiksborgvej 399, P.O. Box 358, 4000
Roskilde, Denmark; E-Mail: [email protected]
* Author to whom correspondence should be addressed; E-Mail:
[email protected]; Tel.: +45-35330856.
Academic Editor: Vronique Martin-Jzquel
Received: 5 February 2015 / Accepted: 28 May 2015 / Published:
16 June 2015
Abstract: Diatoms of the genus Pseudo-nitzschia produce domoic
acid (DA), a toxin that is vectored in the marine food web, thus
causing serious problems for marine organisms and humans. In spite
of this, knowledge of interactions between grazing zooplankton and
diatoms is restricted. In this study, we examined the interactions
between Calanus copepodites and toxin producing Pseudo-nitzschia.
The copepodites were fed with different concentrations of toxic P.
seriata and a strain of P. obtusa that previously was tested to be
non-toxic. The ingestion rates did not differ among the diets (P.
seriata, P. obtusa, a mixture of both species), and they
accumulated 6%16% of ingested DA (up to 420 g per dry weight
copepodite). When P. seriata was exposed to the copepodites, either
through physical contact with the grazers or separated by a
membrane, the toxicity of P. seriata increased (up to 3300%)
suggesting the response to be chemically mediated. The induced
response was also triggered when copepodites grazed on another
diatom, supporting the hypothesis
Mar. Drugs 2015, 13 3810
that the cues originate from the copepodite. Neither pH nor
nutrient concentrations explained the induced DA production.
Unexpectedly, P. obtusa also produced DA when exposed to grazing
copepodites, thus representing the second reported toxic polar
Keywords: Calanus copepodites; Pseudo-nitzschia seriata; P.
obtusa; grazing; induction; chemical ecology; toxin production;
The marine biotoxin domoic acid (DA) causes amnesic shellfish
poisoning (ASP) in humans [1,2], and exposure to DA is known to
have harmful effects on animals in the marine food web, e.g., sea
birds and mammals [3,4]. DA is produced by species of the diatom
genera Nitzschia and Pseudo-nitzschia as a secondary metabolite
[5,6]. DA accumulates in marine organisms that feed on
phytoplankton, e.g., planktivorous fish (such as sardines),
bivalves and copepods , which may then serve as vectors for DA
in the food web. Despite the grim effects that DA-producing diatoms
have on higher trophic levels, only a few studies have explored the
relations between toxic diatoms and their grazers [10,11].
Pseudo-nitzschia is a globally distributed diatom genus, of
which many species form extensive blooms. Fourteen of the 39
described species are known to be toxigenic . The first
recorded ASP incident in 1987 in Prince Edward Island, Canada ,
resulted in increased research interest, and surveillance programs
now monitor concentrations of Pseudo-nitzschia and/or levels of DA
in mollusks. These research and monitoring efforts have
particularly expanded our knowledge of the ecology, distribution,
taxonomy, and toxin-production of Pseudo-nitzschia in temperate,
subtropical and tropical areas. The polar regions, however, have
received much less attention. No records of a toxigenic
Pseudo-nitzschia species exist from the Antarctic and the first,
and so far only, record of a toxin-producing diatom in the Arctic
is P. seriata [12,13].
Several species of copepods are known to graze on toxic
Pseudo-nitzschia. Most studies have not detected any reduction in
grazing on toxic versus non-toxic Pseudo-nitzschia [9,1417]. The
only study conducted on arctic copepods, Calanus spp., did not find
significant differences in the overall weight-specific ingestion of
toxic and non-toxic species in three Calanus species. However,
discontinuous grazing rates were detected, indicating that two of
the species, C. finmarchicus and C. hyperboreus, were temporally
affected when fed with toxic P. seriata. This suggests that DA may
act as a grazing deterrent against copepods . A similar impact
was seen on the grazing pattern in the krill Euphausia pacifica,
when grazing on toxic P. multiseries was compared to non-toxic P.
Pseudo-nitzchia seriata increased production of DA when exposed
to grazing adult Calanus copepods , an effect which may be
related to defense against grazing, and the response was found to
be chemically mediated. Changes in nutrients (silicate, nitrate,
ammonium and phosphate), and changes in pH levels are known
triggers for DA production in Pseudo-nitzschia species .
However, the few studies that have measured changes in nutrients
when investigating induced
Mar. Drugs 2015, 13 3811
responses in phytoplankton by zooplankton, have not found the
nutrients to be the inductive factor .
In the Arctic, copepods of the genus Calanus dominate the
mesozooplankton. Before the phytoplankton spring bloom, the
copepods ascend from the depth to feed, reproduce and spawn .
After spawning, the adult Calanus copepods descend to the water
near the bottom for hibernation . Thereafter, the younger
stages are among the most abundant mesozooplankton and are often
key grazers during the post bloom period [30,31]. The grazing
studies mentioned previously were all performed on adult females.
It is not known if the younger stages graze on Pseudo-nitzschia and
if they do, whether they are more vulnerable to the toxins. To our
knowledge, no other studies have explored the effect of DA on
younger stages of copepods and whether they retain DA, neither have
their effect on toxin production in phytoplankton been
The aims of the present study were to investigate the
interaction between Calanus copepodites and Pseudo-nitzschia, i.e.,
(1) if copepodites select between Pseudo-nitzschia species of
different toxicity or size; (2) if grazing on toxic
Pseudo-nitzschia affects grazing rates and/or mortality of the
copepodites; (3) if copepodites retain DA; (4) if grazing pressure
from the copepodites induces DA production in a toxic (P. seriata)
and a non-toxic (P. obtusa) Pseudo-nitzschia species; and (5) if
the induced DA production is mediated because of changes in the
major inorganic nutrients or pH, or due to waterborne chemical cues
from the copepodites.
2.1. Temporal Grazing Experiment
At the beginning of the experiment, the copepodites and the P.
obtusa cells did not contain DA, whereas P. seriata cells contained
low amounts of DA, ~0.1 pg cell1 (level of detection = 0.003 pg DA
cell1) (Table 1). Domoic acid cell quotas increased significantly
in both P. seriata and P. obtusa when exposed to grazers, but not
in the control without copepodites (Table 1). This is the first
report of DA production by P. obtusa. P. seriata produced more DA
than P. obtusa per cell (t-test, P < 0.001) (Table 1), and also
per volume, i.e., considering the larger biovolume of P. seriata
(~2.2 103 and ~0.5 103 pg DA per m3, for P. seriata and P. obtusa,
respectively) (t-test, P = 0.003). Dissolved DA was not measured.
At the end of the experiment, the copepodites retained 0.1 0.0 ng
DA g C1 after grazing on P. obtusa, the P. obtusa cells produced
0.4 0.1 104 ng DA g C1. After grazing on P. seriata for 39 h, the
copepodites retained 0.6 0.2 ng DA g C1, the P. seriata cells
produced 1.9 4.4 104 ng DA g C1. DA measured in the copepodites
ranged from 8.2 to 69.7 ng DA per individual, with the lowest
amount after grazing on P. obtusa and the highest after grazing on
P. seriata (Mann-Whitney Rank Sum Test, P = 0.03) (Table 1). Of the
ingested DA after grazing on P. obtusa, the copepodites retained
6%, and 13%16% when grazing on P. seriata and mixture of both,
Mar. Drugs 2015, 13 3812
Table 1. Grazing experiment. Pseudo-nitzschia cell density
(cells mL1) and domoic acid (DA) cell quota (pg DA cell1) in P.
seriata and P. obtusa in treatments and controls. Calanus total
ingested (ng DA cop1) and retained DA per copepodite, given in
measured DA (ng DA cop1), by dry weight (g DA g DW cop1) and as
percent of ingested DA. Results are given as * = significant
difference from start to end, and ** for significant difference
between control and treatment. LOD = level of detection. (LOD for
P. obtusa was 0.001 pg DA cell1).
Number of Cells DA Cell Quota Ingested DA Retained DA
(Cells mL1) (pg DA Cell1) (ng DA
(g DA g
Initial End Initial End
P. seriata 3964 40 5048 102 0.1 0.0 0.1 0.0
P. obtusa 5061 32 8907 683
Mar. Drugs 2015, 13 3813
Figure 1. Fecal pellet production and weight-specific ingestion
rate in the 39 h grazing experiment. (a) Fecal pellet production
(cop1 h1); and (b) specific fecal pellet production (SPP) (% h1)
when grazing on P. seriata, P. obtusa or a mixture of both species.
No significant differences were found among treatments; (c)
Weight-specific ingestion rate (% h1), calculated from fecal pellet
production and from clearance were plotted against each other for
the three diets. The weight-specific ingestion rate was
statistically higher when calculated from clearance, than
calculated from fecal pellet egestion.
Mar. Drugs 2015, 13 3814
Figure 2. Grazing in the 39 h experiment. (a) Mean clearance
rate (mL1cop1 h1, mean SD) for grazing on P. seriata, P. obtusa or
a mixture of both species; (b) Weight-specific ingestion rates (%
h1, mean SD) for grazing on P. seriata, P. obtusa or a mixture of
both species. No statistical differences were found among
Mar. Drugs 2015, 13 3815
Fecal pellet production was highest when grazing on P. seriata,
and specific fecal pellet production (SPP) was highest when grazing
on P. obtusa (Figure 1b). However, no significant differences were
found in number of fecal pellets produced (one-way ANOVA, F2,9 =
1.3, P = 0.4) nor in SPP among the diets (one-way ANOVA, F2,9 =
1.0, P = 0.4). Comparison of the weight-specific ingestion rates,
based on clearance of carbon or based on fecal pellet egestion,
showed a higher ingestion rate when based on clearance, than
egestion when grazing on all three diets (t-tests; P. seriata and
mix of both species P < 0.001, and P. obtusa Mann-Whitney Rank
Sum Test, P = 0.03). For both calculation methods, a comparison of
the ingestion rate among the diets showed the highest ingestion
rate on P. obtusa, and the lowest on the mixed diet (Figure
2.1.3. Growth Conditions
The initial cell density in each of the diet treatments was the
same as in the controls (t-test; for P. seriata P = 0.7; P. obtusa
P = 0.1 and in the treatment with mixed cultures: P. seriata P =
0.9 and P. obtusa P = 0.1). Overall, no significant changes or
trends in changes of concentrations of phosphate (PO43), ammonium
(NH4+) and nitrate (NO3) were observed, although single samples
differed either from start to end or between control and treatment
(see details in Supplementary Table S1). Silicate (Si(OH)4)
concentrations decreased significantly in all the controls from
start to end of the experiment, whereas in the treatments with
copepodites there was no significant change. The pH levels were 8.1
0.1 in the controls and the treatments at the start of the
experiment and 8.1 0.03 at the end of the experiment, and no
overall significant changes were observed among the treatments
containing copepodites and the controls (see details in
Supplementary Table S1).
2.2. Induction Experiments
Domoic acid cell quota (toxicity) in P. seriata increased
significantly in both copepodite density treatments, both in flask
A with cells in direct contact with the grazers (Figure 3a), and in
flask B with cells separated from the grazers (Figure 3b) (RM
ANOVA, P-values < 0.05, except for flask A day 2 to 5, where
non-induced cells were added on day 3). Overall, the DA cell quota
gradually increased in flask A from day 0 to day 8, whereas the
response was delayed in flask B (Figure 3). In the control, DA cell
quota did not change during the experiment, it was consistently in
the range 0.3 0.1 to 0.4 0.0 pg DA cell1 (RM ANOVA, F2,6 = 38.8, P
= 0.08). From day 0 to 2, toxicity of P. seriata increased
significantly only in flask A (t-tests, with 12 copepodites P <
0.001 and with 20 P = 0.003). On day 2, the DA cell quota was
significantly higher in flask A than in B (t-tests: P = 0.002 and P
< 0.001 for 12 and 20 copepodites, respectively). After day 2,
toxin content was the same in flask A and B in both concentrations
of copepodites (t-tests, P > 0.05). The highest level of DA was
13.3 4.9 pg DA cell1 in flask A with 12 copepodites on day 8
(Figure 3 and Supplementary Table S2). In the experiment where
cells of P. seriata were grown in filtrate water, where the
copepodites had previously been grazing on Thalassiosira sp., the
increase in DA cell quota was from 0.2 0.0 to 2.4 0.2 pg DA cell1
in 39 h (paired t-test, P = 0.002) (Figure 3c).
Mar. Drugs 2015, 13 3816
Figure 3. Domoic acid cell quota (pg DA cell1, mean SD) of P.
seriata when grazed by two different concentrations of copepodites
and in the control. (a) Flask A with copepodites and P. seriata (n
= 4); (b) Flask B with only P. seriata cells separated from the
copepodites by a 2 m membrane (n = 4). The control contains P.
seriata cells (n = 3). The arrows indicate the time when cells were
added to ensure the copepodites had enough food; (c) Toxicity of P.
seriata (pg DA cell1, mean SD) when grown in the filtrate water
where copepodites had been grazing on another diatom species
(Thalassiosira sp.) Note the different scales in (c).
Mar. Drugs 2015, 13 3817
2.2.2. Growth Rate, Cell Density and Growth Condition
The initial cell concentrations were the same in all treatments
and in the controls at the start of the experiment (one-way ANOVA,
F2,11 = 3.7, P = 0.05). Mean growth rates (eight days) in all
incubators containing only P. seriata cells, i.e., flasks B and
controls, were the same (Kruskal-Wallis test, P = 0.7), indicating
similar growth conditions (Figure 4). The growth rates were
slightly lower during the first two days, 0.1 0.9 d1 and stabilized
thereafter around 0.3 0.1 day1. The cell density was significantly
lower in flask A than in flask B on day 2 in both treatments (12
and 20 copepodites) (Figure 5b) (t-test, P < 0.001 and
Mann-Whitney Rank Sum Test, P = 0.029) illustrating that the
copepodites grazed on the toxic cells. Despite the addition of
cells in flask A on day 3, the cell numbers were significantly
lower in flask A than in flask B on day 8 in both treatments
(Figure 5) (t-test, P = 0.018, and Mann-Whitney Rank Sum Test, P =
Figure 4. Growth rate (day1) in flask B containing only P.
seriata cells, and the controls. On day 3, the flasks were diluted
with 1/10 L medium (arrow) to compensate for adding cells to the
other site of the chamber where the copepodites had reduced the
cell concentration drastically. No statistical differences were
found among treatments.
Mar. Drugs 2015, 13 3818
Figure 5. (a) Cell concentrations (ln cells mL1, mean SD) in the
induction experiment. (a) The two controls (both n = 3); (b) Cell
concentrations in flask A with 12 copepodites and flask B with only
P. seriata cells separated from flask A by a 2 m membrane (n = 4).
(c) Cell concentrations in flask A with 20 copepodites and flask B
with only cells separated from flask A by a 2 m membrane (n = 4).
The cross and arrows indicate the time and the amount for P.
seriata cells or 1/10 L medium addition.
Mar. Drugs 2015, 13 3819
Overall, the nutrient measurements showed a significantly larger
decrease in silicate concentration in the control than in the
treatments, relatively stable concentrations of phosphate and
ammonium throughout the experiment (although single samples
differed either from start to end or between control and treatment
(see details in Table 2)), and a significantly higher end
concentration of nitrate in the treatments than in the control
(Table 2). Because only two measurements were available for the
initial concentration of nitrate, it was not included in the
statistical tests. The initial NO3:Si(OH)4:PO43 ratios were
~23:1:1. The pH levels were 8.1 at the start of the experiment and
varied between 8.1 and 8.2 at the end of the experiment (Table 2),
with no statistical difference between the treatments and the
Table 2. Induction experiments. Nutrient concentrations and pH
levels at the start and the end of the induction experiments. n is
the number of copepoditesr. Values are given as mean SD. Results
are given as * = significant difference between start and end and
** = significant difference between the control and the
(day)/(h) Treatment Si(OH)4 (mol L1) PO43 (mol L1) NH4+ (mol L1)
NO3 (mol L1) pH
0 Initial 5.7 0.6 5.6 0.6 38.3 1.6 131.6 5.6 8.10
8 Control 0.0 * 3.5 0.3 * 25.6 1.0 * 99.3 6.3 8.12 0.02
8 Flask A, n = 12 1.3 1.0 * 6.6 1.2 ** 28.8 2.3 182.6 20.7 **
8 Flask A, n = 20 3.6 0.7 *, ** 7.1 0.5 *, ** 33.9 0.7 ** 191.2
4.2 ** 8.12 0.02
8 Flask B, n = 12 0.4 0.7 * 5.5 1.1 25.9 1.4 164.2 17.7 ** 8.18
8 Flask B, n = 20 0.4 0.3 * 5.1 0.1 27.9 1.5 161.4 10.5 ** 8.11
0 Initial 6.7 0.6 7.0 0.2 38.8 1.7 164.8 12.5 8.06 0.00
39 h Control 3.6 0.3 * 7.4 0.1 * 39.9 1.0 177.7 1.0 8.09
39 h End 3.7 0.31 * 8.0 0.4 * 43.3 0.7 *, ** 197.7 1.1 *, **
2.2.3. Grazing and Retained Domoic Acid
The average clearance rate was 0.7 0.1 mL cop1 h1 in flask A
with 12 copepodites and 1.1 0.2 mL cop1 h1 with 20 copepodites. The
average weight specific ingestion rate (08 days) was the same in
both treatments (0.4% 0.0% h1). The lowest ingestion rate was
measured between days 02 and highest between days 35 after
additional cells had been added (Figure 6). At the end of the
experiment, 106 of the initial 128 copepodites were alive and
vital. In the treatment with 12 copepodites, one was dead, and in
the treatment with 20 copepodites, six copepodites were dead, the
remaining copepodites were not found. After eight days grazing on
P. seriata, the copepodites retained 250 77 and 171 29 ng DA cop1,
in the treatments with 12 and 20 copepodites, respectively,
corresponding to 2.3 0.7 and 1.6 0.3 ng DA g C1. The P. seriata
cells produced on average 8.1 2.5 104 ng DA g C1.
Mar. Drugs 2015, 13 3820
Figure 6. Weight specific ingestion rate of the two
concentrations of grazers (% h1, mean SD). The arrows indicate the
time when cells were added to ensure the copepodites had enough
3. Discussion and Conclusions
3.1. Calanus Copepodites Grazing on Pseudo-nitzschia
In this study we show for the first time that Calanus
copepodites stages C3 and C4 graze on toxic Pseudo-nitzschia and
retain the toxin, suggesting that copepodites pose a risk as
vectors of DA in arctic marine food. The results suggest that DA
did not deter grazing, which may seem as a paradox because the
algae induce toxin production in the presence of grazing
copepodites. But DA may affect other physical capabilities of the
grazer, e.g., the competency to escape predators. This has been
seen in the copepods Oithona similis and C. helgolandicus, which
were negatively affected (disoriented or dead) after feeding on a
toxic strain of Alexandrium tamarense . Other potential effects
of DA could be a reduction in fecundity and hatching rate of eggs,
as has previously been demonstrated for the rotifer Brachionus
plicatilis . The ingestion and clearance rates did not differ
significantly among the treatments or within the treatments, i.e.,
between the light and dark periods, or on average over the 39 h.
The grazing rates were the same in the two experiments and were in
the range found in the other studies on C. finmarchicus copepodites
[34,35]. Our results are in agreement with the results of the
majority of previous grazing studies on adult copepods, which have
not shown DA to deter grazing by copepods, e.g., no selectivity was
observed by  for the copepod Acartia clausi against the
Mar. Drugs 2015, 13 3821
toxic P. multiseries compared to the non-toxic P. delicatissima.
The same was seen for C. finmarchicus grazing on toxic P.
multiseries and non-toxic P. pungens . The results partly
differ from the only study from the same location, where adult
females of C. finmarchicus and C. hyperboreus were temporally
affected when fed with toxic P. seriata , similar to a temporal
grazing effect seen on krill . Deterred grazing and/or
mortality of copepods have been observed in other toxigenic algal
groups, e.g., reduced grazing rates of C. pacificus were detected
when grazing on toxic dinoflagellates [36,37]. Acartia tonsa
ingested lower proportion of toxic Alexandrium minutum when offered
a choice between A. minutum and non-toxic alternative prey
Prorocentrum micans , and grazer mortality ranged from 36% to
47% for Centropages typicus and Acartia clausi, respectively,
grazing on toxic A. minutum . In the present study, both
Pseudo-nitzschia species were, to our surprise, found to be toxic,
and the copepodites fed continuously on the toxic P. seriata as
well as on the less toxic P. obtusa. Hence, due to the lack of a
non-toxic control, we cannot conclude that DA will not have any
negative effects on the copepodites, since, e.g., ingestion rates
might be higher on a non-toxic species than on the toxic
Selectivity for size (grazing differently on P. seriata, which
is larger than P. obtusa) was not observed. This was expected as
the copepodites were in the size range of C. pacificus and C.
finmarchicus, and both P. seriata and P. obtusa are within the size
range of prey of these species [18,39]. When culturing clonal
strains of pennate diatoms, like Pseudo-nitzschia, they inevitably
become shorter with time due to restrictions by the silica encasing
during cell divisionthe MacDonald-Pfitzer rule . The large cell
size is regained during sexual reproduction. The cell length of the
strains of P. seriata and P. obtusa were therefore small compared
to observations of the species in the field. But since the
Pseudo-nitzschia cells appeared in stepped colonies, which the
copepodites apparently did not have difficulties handling, we do
not expect that a larger cell size in the field will pose any
differences in grazing. The copepodites were similar to the size of
an adult C. finmarchicus, which has been reported to feed
effectively on cells with considerable larger volumes than the
cells of the present study , it can therefore be expected that
the feeding response we see is representative for the feeding on
We saw few (six out of 128) dead copepodites in the eight-day
induction experiment, and this could to a minor degree be related
to a hypothesis of DA not acting as grazing deterrent but as a
toxin. As we did not find any effect on grazing as an indication of
the copepodites not being well, we do not consider it likely that
the six dead copepodites had died because of the DA. Dissolved DA
(dDA) has previously been found not to affect the grazing rate but
having lethal effects on the copepod Tigriopus californicus ,
and dDA has been found to increase mortality of krill . dDA was
not measured in the present study, because the same strain had
previously been found to leak DA only in very low amounts in
exponential growth phase , and dDA was thus not considered
relevant. Because most copepodites were alive after grazing on
monocultures of toxic algae, we assume that neither dDA nor
accumulated DA caused copepodite mortality.
The study organisms used here, Pseudo-nitzschia and Calanus,
originate from the same locality, meaning that the Calanus
population may have a long history of exposure to DA. Local
adaptation of copepods to toxins has been studied in Acartia spp.
grazing on toxic dinoflagellates and has shown that populations
with a long history of exposure to toxins show better performance
(ingestion rate and egg production) when exposed to toxic algae, in
comparison to nave populations with no previous
Mar. Drugs 2015, 13 3822
experience of the toxins . If zooplankton populations,
e.g., Calanus copepodites, are adapted to algal toxins, this may
hypothetically, result in a reduced algal cell concentration or
shorter duration of a toxic algal bloom due to grazing, compared to
presence of nave zooplankton (i.e., less reduction in grazing). On
the other hand, zooplankton populations adapted to algal toxins may
accumulate greater quantities of toxins in their bodies, and
therefore pose a larger risk for accumulation of toxins in the food
web and further increased risk of ASP in humans. Something similar
has been observed in the clam Mya arenaria where a variation in
resistance/sensitivity to paralytic shellfish toxins produced by
Alexandrium tamarense was found. Clams from areas exposed to the
toxins were more resistant to the toxins and accumulated the toxins
at higher rates than clams from non-exposed areas, indicating an
adaptation to the toxin .
The production of fecal pellets confirms that the copepodites
were grazing on the algae. The pellet production in this study was
0.3 to 0.4 pellets copepodite1 h1, which is lower than found for C.
finmarchicus copepodites stages C4 and C5 . This can partly be
explained by the accumulation of lipids, which may be higher in
stages C3 and C4 than in C5, as suggested in , thus
illustrating that the younger stages of copepodites are exploiting
the ingested food to a higher degree that the older stages. The
lipids are essential for the overwintering mechanism and early
provision for fueling maturation, gonad development and
reproduction in the spring [51,52]. The high degree of food
exploitation may further explain the lower weight-specific
ingestion rates found when calculations were based on fecal pellets
egestion than on clearance of carbon, as the calculations are based
on assumptions made on adult copepods  (Figure 2c).
3.1.2. Copepodites as Vectors in the Arctic Food Web
This present study is the first conducted on copepodites as
potential vectors for DA in the food chain and the results clearly
show that the copepodites retain DA. The amount of retained DA, in
relation to body weight, ranged from 48 g DA g DW cop1 when grazing
on P. obtusa, to 410 g DA g DW cop1 when grazing on P. seriata, for
39 h. This is in range with the amounts in Tammilehto et al. ,
where the adult Calanus grazed for 12 h on P. seriata and the DA
retained ranged from 68 g DA g DW cop1 in C. glacialis to 290 g DA
g DW cop1 in C. finmarchicus. The body weight of the copepodites
was similar to the size of an adult C. finmarchicus (~0.11 mg C
cop1). The amount of DA retained per copepod, 68 ng DA cop1 when
grazing on P. seriata for 39 h, is in the range of what has
previously been found for adult females; i.e., C. finmarchicus was
found to accumulate 17 5 ng DA cop1 when fed mixed diet of toxic P.
multiseries and non-toxic P. pungens, and 42 4 ng DA cop1 on a diet
of P. multiseries for 12 h . The results are also similar to
the values found in , i.e., 55 10 ng DA cop1 for C.
finmarchicus grazing on P. seriata for 12 h. The percentage of
ingested DA retained in the copepodites ranged from 6% 2% when
grazing in P. obtusa to 16% 4% when grazing on a mixture of both
species. This is slightly lower than for adult Calanus spp. where
values ranged from 37% 32% to 48% 20% in C. hyperboreus and C.
finmarchicus  but in the range of the findings in , 12% 5%
to 34% 19%. These relatively high levels of DA retained in
copepodites clearly illustrate a potential risk for accumulation of
DA in the arctic food web during a toxic bloom of Pseudo-nitzschia.
The amount of DA retained in Calanus copepodites was higher in DA
per g carbon than in the Pseudo-nitzschia cells. Furthermore,
Mar. Drugs 2015, 13 3823
the levels found in the copepodites were higher than amounts
that have been found in krill (
Mar. Drugs 2015, 13 3824
and our assumption is that they partly derive from the
intracellular content of inorganic nutrients released into the
medium when the grazers crush the silicate frustules during
grazing. Copepods may leak ammonium and high levels (>200 M) can
lead to increased cellular DA quota in Pseudo-nitzschia .
Ammonium concentrations were the same in the control and the
treatment, except for higher levels in one treatment (flask B, 20
copepodites) (Table 2), and the levels of ammonium were not in the
range shown to cause increased DA-production . Therefore,
levels of ammonium cannot explain the induced DA production. High
nitrate levels may also trigger DA production [22,59]. Nitrate
concentrations were significantly higher in the treatments than in
the control at the end of the experiment in the induction
experiments. The source of the nitrate most likely originates from
the addition of cells to the A-flasks on day 3. The input of cells
may have resulted in addition of nutrients at higher levels than
the 1/10 medium added to the controls, as the addition was based on
a mixture of 1/10 medium of cells from a full medium culture.
Another possibility is, as mentioned above, the nitrate might
originate from the Pseudo-nitzschia cells when crushed during
grazing. Further, the nitrate might originate from the copepodites
via nitrification occurring on the carapace of the animals, a
phenomenon that is not uncommon among aquatic invertebrates .
The ammonium excretions of the copepodites would be a source of
ammonium for ammonia-oxidizing bacteria . To support this
hypothesis an increase in nitrogen has been detected in incubation
of starving C. hyperboreus from the same locality (Peter Stief,
personal communication) . It should be pointed out that the
levels of nitrate at the start of both the 39 h induction
experiment and the grazing experiment were in range with the end
concentration of the eight-day induction experiment, and as the
latter experiment controls and treatment end concentrations did not
differ in spite of different DA levels, we thus exclude nitrate as
Studies conducted on natural population have demonstrated that
low silicate Si(OH)4:PO4 and Si(OH)4:NO3 ratios correlated with
high DA-production, suggesting silicate limitation causing toxin
production . However, at the same locality,  reported
correlations between low ratios of Si(OH)4:PO43 and (NO3 +
NO22):PO43 correlated with the enhancement in DA production, but
low Si(OH)4:(NO3 + NO22) ratios did not. These studies are partly
in agreement with the results of laboratory results showing that
silica stress increases toxin production in Pseudo-nitzschia. Other
studies have, however, not detected any correlation with ambient
concentrations of the nutrients and DA production [65,66]. Further,
DA production in natural population of Pseudo-nitzschia has been
measured when the levels of nutrient where replete . In the
present study, the nutrient levels of the local seawater were not
available prior to the experiments and not within the Redfield
ratio. In both the treatment and the control, the nutrient levels
were closer to the ratio suggested to elicit DA production;
nonetheless, DA was not induced in the controls. Our findings are
therefore not in agreement with the previous in laboratory
experiments, and the divergence between the field studies further
clearly demonstrates that there is a lack of a strong relationship
between the level of nutrient levels and DA production in the
field. Studies exploring the interaction of factors inducing DA
production are needed to improve our understanding of the factors
inducing DA production in the field.
The cellular toxin levels in the present study were higher than
previously seen in some laboratory experiments with P. seriata,
where e.g., phosphate as the depleting factor resulted in a maximum
of 2.9 pg DA cell1, but were comparable to levels under silicate
depletion were the cells produced max 14.7 pg DA cell1 . The
levels of DA induced during exponential growth phase are within
Mar. Drugs 2015, 13 3825
range seen in P. seriata in the field, e.g. a concentration of
up to 21 pg DA cell1 was reported in  in Danish waters. Similar
concentrations have been measured from Danish, Scottish and
Canadian strains, which yielded DA concentrations of up to 33.6 pg
cell1, 14.7 pg cell1 and 7 pg cell1, respectively [69,70]. The
levels of DA are further in the range found in the field for other
Pseudo-nitzschia species e.g., 0.178 pg cell1 in P. australis ,
775 cell1 in field samples comprising mainly P. australis  and
0117 pg DA cell1 in samples containing a mixture of
Pseudo-nitzschia species .
pH levels in the present study ranged between 8.1 and 8.2 and no
significant differences were found between the start and end of the
experiment or among treatments and controls. Therefore, pH as well
as nutrient concentrations can be excluded as a cause for the
inducing increased DA-production in P. seriata and P. obtusa.
3.3. Toxin Production in the Previously Non-Toxic P. obtusa
Our initial intention was to use P. obtusa as a non-toxic
control strain to explore the effect of toxic diet on the
copepodites. P. obtusa has previously been tested negative for DA
during different growth phases, as well as at pH levels from 8.09.1
[58,73]. Initial tests showed that P. obtusa did not produce DA at
present detection levels, but surprisingly it was revealed to be
toxigenic when induced by copepodites. Whether P. obtusa simply
produced DA at levels below the detection limit and toxin
production was enhanced by the induction, or whether the toxin
production was completely shut down before the induction and the
production was turned on by the induction, cannot be determined in
the present study and needs further attention, as well as the
possibility that other Pseudo-nitzschia species are toxin producing
when exposed to grazers.
3.4. Overall Conclusion
Calanus copepodites stages C3 and C4 grazed on toxic
Pseudo-nitzschia and retained high levels of the toxin, suggesting
that the copepodites are able to tolerate DA and act as vectors of
DA to higher trophic levels, thus posing a threat to the arctic
marine food web and the humans exploiting it. The presence of
grazing copepodites induced DA production in both Pseudo-nitzschia
species, suggesting that production of DA by Pseudo-nitzschia may
be a defense mechanism against grazing, however in this study no
effect were found on the grazing rate. An induced response was also
elicited when the copepodites had previously been grazing on
another diatom species, illustrating that the cues do not originate
from Pseudo-nitzschia cells. The results from both experiments show
that the induced response in production of DA is not caused by
changes in nutrient levels or pH, but suggest that the water borne
cues originate from the copepodites. Finally, the present study is
the first report of P. obtusa being a toxin-producing
Pseudo-nitzschia species, the 15th known DA producer of this
genus.Copepods, including copepodites, constitute an imperative
link, transferring energy from primary producers to higher trophic
levels, in marine food webs and consequences of an increase of
3300% increase in DA production due to grazing of copepodites may
be profound for the marine food web, with further consequences for
human health and economy.
Mar. Drugs 2015, 13 3826
4. Materials and Methods
4.1. Study Organisms
Mesozooplankton was collected from Disko Bay (6914 N, 5323 W),
West Greenland, in June 2013, from the upper 100 m using a WP-2 net
(200 m). Calanus in stages C3 and C4 were identified by the number
of urosome somites and picked individually in petri dishes placed
on ice blocks using stereo microscopes (Nikon SMZ-1B and Leica Wild
M3b). The copepodites were kept in the dark at 4 C for a maximum of
2 weeks in 0.22 m filtered seawater and fed with Thalassiosira sp.
Prior to the experiments, the copepodites were starved for 24 h.
Carbon content of the copepodites (C) was calculated from a
length/weight regression using the equation given for post bloom
period; C = 0.0044 PL3.57, where PL is prosome length (Table 3).
Dry weight was calculated assuming the carbon weight to dry mass
ratio to be 0.60 .
Table 3. Size and carbon content of the study organisms. Cell
length and width (m), cell volume (m3), and carbon content (pg C
cell1) for Pseudo-nitzschia obtusa and P. seriata. Length promosome
(mm) and carbon weight of the copepodites, n is the number of
cells/individuals measured and values are given in mean SD.
Pseudo-nitzschia Cell Length (m) Cell Width (m) Cell Volume (m3)
Carbon Content (pg C cell1)P. obtusa
n = 23 33.5 3.2 4.2 0.5 489 149 57.3
P. seriata n = 23
49.0 3.4 5.4 0.8 1163 383 137.1
Calanus Prosome length (mm) Carbon content per
individual (mg cop1) n = 21 2.5 0.5 0.11 0.1
For calculations, phytoplankton growth rate in the controls was
calculated using an exponential model and changes in phytoplankton
concentrations in the grazing were calculated after . Ingestion
rate was calculated as number of food items consumed by each grazer
and weight-specific ingestion was estimated by multiplying number
of Pseudo-nitzschia cells ingested by the cellular carbon content
and dividing it by the average carbon body weight of copepodites.
For the equations in detail see .
At the end of the experiment, fecal pellets were counted and the
size estimated. The width of >20 fecal pellets per treatment and
the length >30 fecal pellets per replicate were measured, and
only pellets that were at least 3 longer than wide were included.
Fecal pellet volume was calculated assuming a cylinder shape and
the carbon content was estimated by applying a volume to a carbon
conversion factor of 0.043 pg C m3 . Specific fecal pellet
production (SPP) was calculated according to  using number of
fecal pellets, the average volume of fecal pellets and the pellet
volume to carbon conversion factor. Weight-specific ingestion was
calculated from fecal pellet production, i.e., egestion and
assimilation efficiency following , and the assimilation
efficiency was assumed to be 0.65 after . All calculations were
conducted following the equations as they appear in .
Mar. Drugs 2015, 13 3827
Strains of P. seriata (strain P5G3) and P. obtusa (strain L4B4)
were isolated into clonal cultures from Disko Bay (6914 N, 5323 W),
in April 2010 and in April 2012, respectively, by isolating single
cells or chains into micro well plates, and later inoculating the
cultures into tissue culture flasks. Strain P5G3 was known to be a
toxic strain of P. seriata, whereas P. obtusa was previously only
known as a non-toxic species . Strain P5G3 is the same strain
as used in the previous experiments on Calanus and Pseudo-nitzschia
in Disko Bay by Tammilehto et al. [18,20]. Prior to the
experiments, both species were grown in batch cultures at 4 C in
L1-medium  under a light intensity of 85 mol photons m2s1, at a
18:6 light:dark cycle using cool white fluorescent bulbs. Carbon
cell quota was calculated using measured carbon content of P.
multiseries (0.12 pg C m3) following  and relating this to the
cell volume of P. seriata and P. obtusa. The cell volume of
Pseudo-nitzschia spp. was calculated following : volume = (0.6
L W2) + (0.4 0.5 L W2), where L is the cell length and W is the
cell width (Table 3).
4.2. Experiment Preparation, Nutrient and DA Analyses
Actively swimming copepodites were sorted out in culture flasks
filled with 0.22 m filtered seawater. Triplicate subsamples of
copepodites were taken for DA analysis, where each replicate
contained the same number of specimens as used in the experimental
flasks. The copepodites were rinsed and filtered onto GF/F filters
using gentle vacuum, and frozen at 20 C until toxin analysis.
Targeted initial diatom cell concentrations were prepared by
diluting the cultures of P. seriata and P. obtusa with an
appropriate volume of 1/10 L1-medium. The initial cell
concentrations provided copepodites with saturating carbon supply
(>400 g C L1) . To avoid artifacts from extreme nutrient
concentrations, 1/10 L1 medium was used, based on 0.22 m filtered
seawater with a salinity of 36. Additional ammonium chloride (0.1
mL of 500 mM NH4Cl L1) was added to the medium to avoid large
differences in ammonium concentrations among treatments due to
ammonium excreted by copepods . Local seawater was used for the
experiments, and we were unfortunately not able to measure nutrient
levels before performing the experiment, and therefore the initial
levels of silicate and phosphate were lower than anticipated. pH
was measured using a WTW pH 3110 pH-meter with a SenTix 41
electrode (a sensor detection limit of 0.01; two point calibration)
(WTW, Xylem), and triplicate subsamples of 50 mL for inorganic
nutrient composition were taken. For measuring DA cell quota,
triplicate subsamples of 200 mL P. seriata and P. obtusa were taken
from the final culture for the experiments, and filtered onto GF/F
filters. Nutrient samples and samples for DA analysis were stored
at 20 C until analysis. The nutrients were analyzed at the
Institute for Bioscience, Aarhus University in Denmark on a flow
injection auto-analyzer, following . DA analyses were conducted
by liquid chromatography coupled with tandem mass spectrometry as
described in details in . Detection limit for DA was 1 ng
sample1. All experiments were run at a temperature of 4 C, and a
light intensity of 100 mol photons m2s1 and 12:12 light:dark cycle
using cool white fluorescent bulbs. The experimental flasks were
mounted on a plankton wheel with speed 1.3 rpm.
Mar. Drugs 2015, 13 3828
4.3. Grazing Experiment
The copepodites were fed with cultures of toxic P. seriata,
non-toxic P. obtusa, and a mixture of both species. Each of the
three grazing experimental treatments were carried out in 720 mL
polystyrene flasks (Sarstedt) in four replicates with 12
copepodites per flask and run in parallel with controls in 3
replicates for 39 h. The nominal initial cell concentration was
4000 cells mL1 of P. seriata and 5000 cells mL1 of P. obtusa, and
2000 cells mL1 of P. seriata plus 5000 cells mL1 of P. obtusa in
the mixed culture for both treatments and controls (Table 3).
At the beginning and at the end of the experiment, samples for
nutrients and DA as well as pH measurements were done as described
in Section 4.4. Subsamples for enumerating cell concentrations were
taken every 3 h, and the volume removed was replaced with 0.22 m
filtered seawater. At the end of the experiment (39 h), the
copepodites and fecal pellets were collected as described in
4.4. Induction Experiment
Induction experiment was carried out in specially designed
incubators, made of two 720 mL polystyrene tissue culture flasks
connected via two apertures (4.5 cm in diameter) with a 2 m
polycarbonate membrane, see details in Tammilehto et al. . The
transmission of food dye through the membrane was measured on a
synergy scanner (Biotek), and the diffusion equilibrium was ~30%
after two days, matching the diffusion over a similar membrane in a
set up made by Tang et al. . The membrane allowed water
exchange between the flasks but the plankton remained in their
initial flasks, named A and B for the two sides. Cells of P.
seriata and copepodites in concentrations of either 12 or 20
individuals were inoculated into flask A, and only P. seriata into
flask B. The initial cell concentration of P. seriata was 3000
cells mL1. The experiment was run in 4 replicates and a control in
3 replicates (using single flasks, 720 mL) for eight days.
Subsamples for cell counts (2 mL; fixed with 2% (final
concentration) acidic Lugols solution) and DA analyses (200 mL)
were taken on days 0, 2, 5 and 8. The volume sampled was replaced
with 0.22 m filtered seawater. On day 3, 168 mL of P. seriata
culture of approximately 7000 cells mL1 was added into flask A to
supplement for a substantial amount of cells grazed by the
copepodites and to ensure the copepodites a sufficient amount of
cells for grazing. Similarly, 168 mL of 1/10 L1 medium was added to
the controls and flask B. New controls in three replicates were
subsequently established for measuring growth rate of the cells
that had been added to the treatments. The cells originated from a
culture based on a mixture of 1/10 L1 medium and cells grown in
full medium. At the end of the experiment (day 8), pH was measured
and subsamples for cell enumeration, nutrients and DA analyses were
taken as described above. The copepodites were collected by sieving
the total content through a 200 m sieve to remove phytoplankton and
fecal pellets. The copepodites were rinsed with 0.22 m filtered
seawater, and individually transferred to a GF/F filter and stored
at 20 C.
4.5. Induction Experiment in Filtrate Water
Filtrate water was prepared by placing >70 actively swimming
copepodites into 0.22 m (~6 L) filtered seawater with a unialgal
culture of Thalassiosira sp. for 24 h. The copepodites and the
fecal pellets were removed by using a 50 m sieve and the cells of
Thalassiosira sp. removed by filtering
Mar. Drugs 2015, 13 3829
through a 0.22 m filter. Experimental flasks with an initial
cell concentration of 4000 cells mL1 of P. seriata, was prepared
using this filtrated water using 720 mL polystyrene flasks. The
experiment was conducted in four replicates and three controls and
run on the plankton wheel simultaneously for 39 h with the grazing
experiment (Section 4.3).
For enumeration of the algal cells, a Sedgewick-Rafter chamber
and an inverted light microscope (Olympus CKX31 at a 100
magnification). A minimum of 400 cells were counted. Fecal pellets
were counted using a stereo microscope (Reichter at 16
magnification) and width length of the fecal pellets was measured
using 40 magnification. For measurements of Calanus and
Pseudo-nitzschia see Sections 4.1.1 and 4.1.2.
4.7. Statistical Analyses
Normal distribution of data was tested using a Shapiro-Wilk test
and homogeneity of variances applying Levenes test. The differences
over time within each treatment were tested by repeated-measures
ANOVA (RM ANOVA) and post hoc analysis tested using Holms-Sidak
adjustments. Differences between means were tested using one-way
ANOVA, a t-test or a paired t-test. If the data set did not pass
the assumption of normal distribution, a Mann-Whitney U-test or a
Kruskall-Wallis test were used. For the nutrients measurements in
Table 2 and Supplementary Table S1, the statistical analyses were
conducted with paired t-test between the start and end, and t-test
between the control end and the treatments end. Results given as *
= significant difference from control start an ** for significant
difference from the control end. The level of significance used was
We thank the Arctic station in Qeqertarsuaq, Greenland and the
scientific leader Ole Stecher for providing excellent research
facilities and help in many ways. We thank Abel Brand and Johannes
Mlgaard for assistance with collecting samples, Lis Munk
Frederiksen for fieldwork and assistance with the experiments. We
thank Wolfgang Drebing for sample extraction and toxin measurements
and Dorottya Sarolta Wgner for assisting with spectrophotometric
measurements. Funding was provided by the Carlsberg Foundation
(2012_01_0556) and grant DFF1323-00258 from the Danish Research
Council to NL and a grant given from: Botanisk forening; Konto af
1988 for botaniske mikroorganismeforskning to SH.
Sara Harardttir: Designed the study, carried out the
experiments, enumerated the samples, analysed the data and wrote
Marina Pani: Carried out the experiments, enumerated the
samples, analysed the data and revised the manuscript.
Anna Tammilehto: Designed the study, discussed the results and
revised the manuscript. Bernd Krock: Performed toxin analyses and
revised the manuscript.
Mar. Drugs 2015, 13 3830
Eva Friis Mller: Performed the nutrient analyses and revised the
manuscript. Torkel Gissel Nielsen: Discussed the results, revised
the manuscript. Nina Lundholm: Designed the study, carried out the
experiments, discussed the results and revised
Conflicts of Interest
The authors declare no conflict of interest.
1. Quilliam, M.A.; Wright, J.L.C. The amnesic shellfish
poisoning mystery. Anal. Chem. 1989, 61, 1053A1060A.
2. Perl, T.M.; Bedard, L.; Kosatsky, T.; Hockin, J.C.; Todd,
E.C.D.; Remis, R.S. An outbreak of toxic encephalopathy caused by
eating mussels contaminated with domoic acid. N. Engl. J. Med.
1990, 322, 17751780.
3. Fritz, L.; Quilliam, M.A.; Wright, J.L.C.; Beale, A.M.; Work,
T.M. An outbreak of domoic acid poisoning attributed to the pennate
diatom Pseudo-nitzschia australis. J. Phycol. 1992, 28, 439442.
4. Fire, S.E.; Wang, Z.H.; Berman, M.; Langlois, G.W.; Morton,
S.L.; Sekula-Wood, E.; Benitez-Nelson, C.R. Trophic transfer of the
harmful algal toxin domoic acid as a cause of death in a Minke
whale (Balaenoptera acutorostrata) stranding in Southern
California. Aquat. Mamm. 2010, 36, 342350.
5. Trainer, V.L.; Bates, S.S.; Lundholm, N.; Thessen, A.E.;
Cochlan, W.P.; Adams, N.G.; Trick, C.G. Pseudo-nitzschia
physiological ecology, phylogeny, toxicity, monitoring and impacts
on ecosystem health. Harmful Algae 2012, 14, 271300.
6. Smida, D.B.; Lundholm, N.; Kooistra, W.; Sahraoui, I.;
Ruggiero, M.V.; Kotaki, Y.; Ellegaard, M.; Lambert, C.; Mabrouk,
H.H.; Hlaili, A.S. Morphology and molecular phylogeny of Nitzschia
bizertensis sp. Nov. A new domoic acid producer. Harmful Algae
2014, 32, 4963.
7. Costa, P.R.; Garrido, S. Domoic acid accumulation in the
sardine Sardina pilchardus and its relationship to Pseudo-nitzschia
diatom ingestion. Mar. Ecol. Prog. Ser. 2004, 284, 261268.
8. Wohlgeschaffen, G.D.; Mann, K.H.; Rao, D.V.S.; Pocklington,
R. Dynamics of the phycotoxin domoic acid accumulation and
excretion in 2 commercially important bivalves. J. Appl. Phycol.
1992, 4, 297310.
9. Maneiro, I.; Iglesias, P.; Guisande, C.; Riveiro, I.;
Barreiro, A.; Zervoudaki, S.; Graneli, E. Fate of domoic acid
ingested by the copepod Acartia clausi. Mar. Biol. 2005, 148,
10. Turner, J.T. Planktonic marine copepods and harmful algae.
Harmful Algae 2014, 32, 8193. 11. Lelong, A.; Hegaret, H.; Soudant,
P.; Bates, S.S. Pseudo-nitzschia (Bacillariophyceae) species,
domoic acid and amnesic shellfish poisoning: Revisiting previous
paradigms. Phycologia 2012, 51, 168216.
12. Hansen, L.R.; Soylu, S..; Kotaki, Y.; Moestrup, .; Lundholm,
N. Toxin production and temperature-induced morphological variation
of the diatom Pseudo-nitzschia seriata from the Arctic. Harmful
Algae 2011, 10, 689696.
Mar. Drugs 2015, 13 3831
13. Lundholm, N.; Skov, J.; Pocklington, R.; Moestrup, . Domoic
acid, the toxic amino-acid responsible for amnesic shellfish
poisoning, now in Pseudonitzschia seriata (Bacillariophyceae) in
Europe. Phycologia 1994, 33, 475478.
14. Lincoln, J.A.; Turner, J.T.; Bates, S.S.; Leger, C.;
Gauthier, D.A. Feeding, egg production, and egg hatching success of
the copepods Acartia tonsa and Temora longicornis on diets of the
toxic diatom Pseudo-nitzschia multiseries and the non-toxic diatom
Pseudo-nitzschia pungens. Hydrobiologia 2001, 453, 107120.
15. Leandro, L.F.; Teegarden, G.J.; Roth, P.B.; Wang, Z.;
Doucette, G.J. The copepod Calanus finmarchicus: A potential vector
for trophic transfer of the marine algal biotoxin, domoic acid. J.
Exp. Mar. Biol. Ecol. 2010, 382, 8895.
16. Tester, P.A.; Pan, Y.; Douchette, G.J. Accumulation of
domoic acid activity in copepods. In Harmful and Toxic Algal Blooms
2000; Hallegraeff, G.M., Blackburn, S.I., Bolch, C.J., Lewis, R.J.,
Eds.; Intergovernmental Oceanographic Commission of UNESCO: Paris,
France, 2001; pp. 401404.
17. Windust, A. The Responses of Bacteria, Microalgae, and
Zooplankton to the Diatom Nitzschia pungens f. Multiseries and Its
Toxic Metabolite Domoic Acid. M.S. Thesis, Dalhousie University,
Halifax, Canada, 1992.
18. Tammilehto, A.; Nielsen, T.G.; Krock, B.; Mller, E.F.;
Lundholm, N. Calanus spp.-vectors for the biotoxin, domoic acid, in
the Arctic marine ecosystem? Harmful Algae 2012, 20, 165174.
19. Bargu, S.; Marinovic, B.; Mansergh, S.; Silver, M.W. Feeding
responses of krill to the toxin-producing diatom Pseudo-nitzschia.
J. Exp. Mar. Biol. Ecol. 2003, 284, 87104.
20. Tammilehto, A.; Nielsen, T.G.; Krock, B.; Mller, E.F.;
Lundholm, N. Induction of domoic acid production in the toxic
diatom Pseudo-nitzschia seriata by calanoid copepods. Aquat.
Toxicol. 2015, 159, 5261.
21. Fehling, J.; Davidson, K.; Bolch, C.J.; Bates, S.S. Growth
and domoic acid production by Pseudo-nitzschia seriata
(Bacillariophyceae) under phosphate and silicate limitation. J.
Phycol. 2004, 40, 674683.
22. Bates, S.S.; Worms, J.; Smith, J.C. Effects of ammonium and
nitrate on growth and domoic acid production by Nitzschia pungens
in batch culture. Can. J. Fish. Aquat. Sci. 1993, 50, 12481254.
23. Lundholm, N.; Hansen, P.J.; Kotaki, Y. Effect of pH on
growth and domoic acid production by potentially toxic diatoms of
the genera Pseudo-nitzschia and Nitzschia. Mar. Ecol. Prog. Ser.
2004, 273, 115.
24. Selander, E.; Thor, P.; Toth, G.; Pavia, H. Copepods induce
paralytic shellfish toxin production in marine dinoflagellates.
Proc. R. Soc. B 2006, 273, 16731680.
25. Selander, E.; Cervin, G.; Pavia, H. Effects of nitrate and
phosphate on grazer-induced toxin production in Alexandrium
minutum. Limnol. Oceanogr. 2008, 53, 523530.
26. Long, J.D.; Smalley, G.W.; Barsby, T.; Anderson, J.T.; Hay,
M.E. Chemical cues induce consumer-specific defenses in a
bloom-forming marine phytoplankton. Proc. Natl. Acad. Sci. USA
2007, 104, 1051210517.
27. Lundgren, V.; Granli, E. Grazer-induced defence in
Phaeocystis globosa (Prymnesiophyceae): Influence of different
nutrient conditions. Limnol. Oceanogr. 2010, 55, 19651976.
Mar. Drugs 2015, 13 3832
28. Tande, K.S. Ecological investigations on the zooplankton
community of Balsfjorden, northern Norway: Generation cycles, and
variations in body-weight and body content of carbon and nitrogen
related to overwintering and reproduction in the copepod Calanus
finmarchicus (Gunnerus). J. Exp. Mar. Biol. Ecol. 1982, 62,
29. Hansen, B.W.; Nielsen, T.G.; Levinsen, H. Plankton community
structure and carbon cycling on the western coast of Greenland
during the stratified summer situation. III. Mesozooplankton.
Aquat. Microb. Ecol. 1999, 16, 233249.
30. Nielsen, T.G.; Hansen, B. Plankton community structure and
carbon cycling on the western coast of Greenland during and after
the sedimentation of a diatom bloom. Mar. Ecol. Prog. Ser. 1995,
31. Madsen, S.D.; Nielsen, T.G.; Hansen, B.W. Annual population
development and production by Calanus finmarchicus, C. glacialis
and C. hyperboreus in Disko bay, western Greenland. Mar. Biol.
2001, 139, 7593.
32. Wohlrab, S.; Iversen, M.H.; John, U. A molecular and
co-evolutionary context for grazer induced toxin production in
Alexandrium tamarense. PLoS ONE 2010, 5, e15039.
33. Whyte, J.N.C.; Townsend, L.D.; Ginther, N.G. Fecundity,
toxin and trophic levels of the rotifer Brachionus plicatilis fed
Pseudo-nitzschia pungens f. Multiseries. Available online:
http://diatom. myspecies.info/node/331 (accessed on 23 May
34. Meyer, B.; Irigoien, X.; Graeve, M.; Head, R.N.; Harris,
R.P. Feeding rates and selectivity among nauplii, copepodites and
adult females of Calanus finmarchicus and Calanus helgolandicus.
Helgoland Mar. Res. 2002, 56, 169176.
35. Koski, M.; Wexels Riser, C. Post-bloom feeding of Calanus
finmarchicus copepodites: Selection for autotrophic versus
heterotrophic prey. Mar. Biol. Res. 2006, 2, 109119.
36. Huntley, M.; Sykes, P.; Rohan, S.; Marin, V.
Chemically-mediated rejection of dinoflagellate prey by the
copepods Calanus pacificus and Paracalanus parvusmechanism,
occurrence and significance. Mar. Ecol. Prog. Ser. 1986, 28,
37. Sykes, P.F.; Huntley, M.E. Acute physiological reactions of
Calanus pacificus to selected dinoflagellatesdirect observations.
Mar. Biol. 1987, 94, 1924.
38. Bergkvist, J.; Selander, E.; Pavia, H. Induction of toxin
production in dinoflagellates: The grazer makes a difference.
Oecologia 2008, 156, 147154.
39. Frost, B.W. Effects of size and concentration of food
particles on feeding behavior of marine planktonic copepod Calanus
pacificus. Limnol. Oceanogr. 1972, 17, 805815.
40. Round, F.E.; Crawford, R.M.; Mann, D.G. The Diatoms: Biology
& Morphology of the Genera; Cambridge University Press: London,
41. Levinsen, H.; Turner, J.T.; Nielsen, T.G.; Hansen, B.W. On
the trophic coupling between protists and copepods in arctic marine
ecosystems. Mar. Ecol. Prog. Ser. 2000, 204, 6577.
42. Shaw, B.A.; Andersen, R.J.; Harrison, P.J. Feeding deterrent
and toxicity effects of apo-fucoxanthinoids and phycotoxins on a
marine copepod (Tigriopus californicus). Mar. Biol. 1997, 128,
43. Bargu, S.; Lefebvre, K.; Silver, M.W. Effect of dissolved
domoic acid on the grazing rate of krill Euphausia pacifica. Mar.
Ecol. Prog. Ser. 2006, 312, 169175.
Mar. Drugs 2015, 13 3833
44. Colin, S.P.; Dam, H.G. Comparison of the functional and
numerical responses of resistant versus non-resistant populations
of the copepod Acartia hudsonica fed the toxic dinofiagellate
Alexandrium tamarense. Harmful Algae 2007, 6, 875882.
45. Colin, S.P.; Dam, H.G. Latitudinal differentiation in the
effects of the toxic dinoflagellate Alexandrium spp. On the feeding
and reproduction of populations of the copepod Acartia hudsonica.
Harmful Algae 2002, 1, 113125.
46. Colin, S.P.; Dam, H.G. Testing for resistance of pelagic
marine copepods to a toxic dinoflagellate. Evol. Ecol. 2004, 18,
47. Jiang, X.; Lonsdale, D.J.; Gobler, C.J. Rapid gain and loss
of evolutionary resistance to the harmful dinoflagellate
Cochlodinium polykrikoides in the copepod Acartia tonsa. Limnol.
Oceanogr. 2011, 56, 947954.
48. Zheng, Y.; Dam, H.G.; Avery, D.E. Differential responses of
populations of the copepod Acartia hudsonica to toxic and
nutritionally insufficient food algae. Harmful Algae 2011, 10,
49. Bricelj, V.M.; Connell, L.; Konoki, K.; MacQuarrie, S.P.;
Scheuer, T.; Catterall, W.A.; Trainer, V.L. Sodium channel mutation
leading to saxitoxin resistance in clams increases risk of PSP.
Nature 2005, 434, 763767.
50. Hygum, B.H.; Rey, C.; Hansen, B.W.; Tande, K. Importance of
food quantity to structural growth rate and neutral lipid reserves
accumulated in Calanus finmarchicus. Mar. Biol. 2000, 136,
51. Jnasdttir, S.H. Lipid content of Calanus finmarchicus during
overwintering in the Faroe-Shetland Channel. Fish. Oceanogr. 1999,
52. Niehoff, B. The effect of food limitation on gonad
development and egg production of the planktonic copepod Calanus
finmarchicus. J. Exp. Mar. Biol. Ecol. 2004, 307, 237259.
53. Cushing, D.H.; Humphrey, G.F.; Banse, K.; Laevastu, T.
Report of the committee on terms and equivalents. Rapp. P.-V. Reun.
Cons. Int. Explor. Mer. 1958, 144, 1516.
54. Swalethorp, R.; Kjellerup, S.; Dnweber, M.; Nielsen, T.G.;
Mller, E.F.; Rysgaard, S.; Hansen, B.W. Grazing, egg production,
and biochemical evidence of differences in the life strategies of
Calanus finmarchicus, C. glacialis and C. hyperboreus in Disko bay,
western Greenland. Mar. Ecol. Prog. Ser. 2011, 429, 125144.
55. Harris, R.P.; Wiebe, P.H.; Lenz, J.; Skjoldal, H.R.;
Huntley, M. Zooplankton Methodology Manual; Elsevier Academic
Press: Beijing, China, 2005.
56. Thor, P.; Wendt, I. Functional response of carbon absorption
efficiency in the pelagic calanoid copepod Acartia tonsa Dana.
Limnol. Oceanogr. 2010, 55, 17791789.
57. Selander, E.; Kubanek, J.; Hamberg, M.; Andersson, M.X.;
Cervin, G.; Pavia, H. Predator lipids induce paralytic shellfish
toxins in bloom-forming algae. Proc. Natl. Acad. Sci. USA 2015,
58. Trimborn, S.; Lundholm, N.; Thoms, S.; Richter, K.U.; Krock,
B.; Hansen, P.J.; Rost, B. Inorganic carbon acquisition in
potentially toxic and non-toxic diatoms: The effect of pH-induced
changes in seawater carbonate chemistry. Physiol. Plant. 2008, 133,
59. Howard, M.D.A.; Cochlan, W.P.; Ladizinsky, N.; Kudela, R.M.
Nitrogenous preference of toxigenic Pseudo-nitzschia australis
(Bacillariophyceae) from field and laboratory experiments. Harmful
Algae 2007, 6, 206217.
Mar. Drugs 2015, 13 3834
60. Heisterkamp, I.M.; Schramm, A.; de Beer, D.; Stief, P.
Nitrous oxide production associated with coastal marine
invertebrates. Mar. Ecol. Prog. Ser. 2010, 415, 19.
61. Koops, H.-P.; Purkhold, U.; Pommerening-Roeser, A.;
Timmermann, G.; Wagner, M. The Lithoautotrophic Ammonia-Oxidizing
Bacteria; Springer: New York, NY, USA, 2006; pp. 778811.
62. Stief, P. University of Southern Denmark, Odense, Denmark.
Personal communication, 2015. 63. Anderson, C.R.; Brzezinski, M.A.;
Washburn, L.; Kudela, R. Circulation and environmental
conditions during a toxigenic Pseudo-nitzschia australis bloom
in the Santa Barbara Channel, california. Mar. Ecol. Prog. Ser.
2006, 327, 119133.
64. Schnetzer, A.; Miller, P.E.; Schaffner, R.A.; Stauffer,
B.A.; Jones, B.H.; Weisberg, S.B.; DiGiacomo, P.M.; Berelson, W.M.;
Caron, D.A. Blooms of Pseudo-nitzschia and domoic acid in the San
Pedro Channel and Los Angeles harbor areas of the Southern
California Bight, 20032004. Harmful Algae 2007, 6, 372387.
65. Trainer, V.L.; Hickey, B.M.; Lessard, E.J.; Cochlan, W.P.;
Trick, C.G.; Wells, M.L.; MacFadyen, A.; Moore, S.K. Variability of
Pseudo-nitzschia and domoic acid in the Juan de Fuca eddy region
and its adjacent shelves. Limnol. Oceanogr. 2009, 54, 289308.
66. Trainer, V.L.; Wells, M.L.; Cochlan, W.P.; Trick, C.G.;
Bill, B.D.; Baugh, K.A.; Beall, B.F.; Herndon, J.; Lundholm, N. An
ecological study of a massive bloom of toxigenic Pseudo-nitzschia
cuspidata off the Washington state coast. Limnol. Oceanogr. 2009,
67. Marchetti, A.; Trainer, V.L.; Harrison, P.J. Environmental
conditions and phytoplankton dynamics associated with
Pseudo-nitzschia abundance and domoic acid in the Juan de Fuca
eddy. Mar. Ecol. Prog. Ser. 2004, 281, 112.
68. Lundholm, N.; Andersen, P.; Jorgensen, K.; Thorbjornsen,
B.R.; Cembella, A.; Krock, B. Domoic acid in danish blue mussels
due to a bloom of Pseudo-nitzschia seriata. Harmful Algae News
2005, 29, 810.
69. Fehling, J.; Green, D.H.; Davidson, K.; Bolch, C.J.; Bates,
S.S. Domoic acid production by Pseudo-nitzschia seriata
(Bacillariophyceae) in Scottish waters. J. Phycol. 2004, 40,
70. Bates, S.S.; Lger, C.; White, J.M.; MacNair, N.; Ehrman,
J.M.; Levasseur, M.; Couture, J.Y.; Gagnon, R.; Bonneau, F.;
Michaud, S.; et al. Pennate diatom Pseudo-nitzschia seriata; domoic
acid production causes spring closures of shellfish harvesting for
the first time in the southern gulf of St. Lawrence, eastern
Canada. In Proceedings of the 17th International Diatom Symposium,
Ottawa, Canada, 2531 August 2002.
71. Trainer, V.L.; Adams, N.G.; Bill, B.D.; Stehr, C.M.; Wekell,
J.C.; Moeller, P.; Busman, M.; Woodruff, D. Domoic acid production
near California coastal upwelling zones, June 1998. Limnol.
Oceanogr. 2000, 45, 18181833.
72. Scholin, C.A.; Gulland, F.; Doucette, G.J.; Benson, S.;
Busman, M.; Chavez, F.P.; Cordaro, J.; DeLong, R.; De Vogelaere,
A.; Harvey, J.; et al. Mortality of sea lions along the central
California coast linked to a toxic diatom bloom. Nature 2000, 403,
73. Hasle, G.R.; Lundholm, N. Pseudo-nitzschia seriata f. obtusa
(Bacillariophyceae) raised in rank based on morphological,
phylogenetic and distributional data. Phycologia 2005, 44,
74. Guillard, R.R.L.; Hargraves, P.E. Stichochrysis immobilis is
a diatom, not a chyrsophyte. Phycologia 1993, 32, 234236.
Mar. Drugs 2015, 13 3835
75. Hansen, P.J.; Bjornsen, P.K.; Hansen, B.W.; Bjrnsen, P.K.
Zooplankton grazing and growth: Scaling within the 22000 m body
size range. Limnol. Oceanogr. 1997, 42, 687704.
76. Corner, E.D.S.; Newell, B.S. On nutrition and metabolism of
zooplankton 4 forms of nitrogen excreted by Calanus. J. Mar. Biol.
Assoc. UK 1967, 47, 113120.
77. Hansen, H.P.; Koroleff, F. Determination of nutrients. In
Methods of Seawater Analysis; Wiley-VCH Verlag GmbH: Weinheim,
Germany, 2007; pp. 159228.
78. Tang, K.W. Grazing and colony size development in
Phaeocystis globosa (Prymnesiophyceae): The role of a chemical
signal. J. Plankton Res. 2003, 25, 831842.
2015 by the authors; licensee MDPI, Basel, Switzerland. This
article is an open access article distributed under the terms and
conditions of the Creative Commons Attribution license